Amphoteric liposomes and their use

ABSTRACT

Amphoteric liposomes are proposed, which comprise positive and negative membrane-based or membrane-forming charge carriers as well as the use of these liposomes.

[0001] The invention relates to amphoteric liposomes, whichsimultaneously comprise positive and negative membrane-based ormembrane-forming charge carriers as well as to the use of theseliposomes.

[0002] The concept of lipids comprises three classes of naturalproducts, which can be isolated from biological membranes:phospholipids, sphingolipids and cholesterol with its derivatives.However, it also comprises synthetically produced materials with similarcharacteristics. As representatives of these, diacyl glycerols, dialkylglycerols, 3-amino-1,2-dihydroxypropane esters or ethers and alsoN,N-dialkylamines are mentioned.

[0003] These substances are of technical interest for the preparation ofliposomes. One of the uses of these liposomes is as a container foractive ingredients in pharmaceutical preparations. For this purpose, anefficient and stable packaging of the cargo, compatibility with bodyfluids and a controllable and optionally site-specific release of thecontent are desirable.

[0004] It is a disadvantage that it is difficult to combine the tworequirements. The tighter and more stable the packaging, the moredifficult it is to release the enclosed active ingredient once again.For this reason, liposomes were developed, which change their propertiesin reaction to external stimuli. Heat-sensitive and pH-sensitiveliposomes are known. The pH-sensitive liposomes are of special interest,since this parameter may change under physiological circumstances, suchas during the endocytotic absorption of a liposome in cells or duringpassage through the gastrointestinal tract. According to the state ofthe art, pH-sensitive liposomes comprise, in particular, cholesterolhemisuccinate (CHEMS).

[0005] Cholesterol hemisuccinate is used in admixture with phosphatidylethanolamine for the preparation of pH-sensitive liposomes (Tachibana etal. (1998); BBRC 251:538-544, U.S. Pat. No. 4,891,208). Such liposomescan be endocytized by many cells and in this way are able to transportcargo molecules into the interior of cells, without injuring theintegrity of the cellular membrane.

[0006] The anionic character of CHEMS is a significant disadvantage. Theliposomes, prepared with it, have an overall negative charge andabsorbed by cells only with a low efficiency. Therefore, in spite of thetransfer mechanism described above, they are hardly suitable fortransporting macromolecules into cells.

[0007] Cationic liposomes with the highest possible and constant surfacecharge are used to transport active ingredients into cells(transfection). The overall positive charge of such particles leads toan electrostatic adhesion to cells and, consequently, to an efficienttransport into cells. The use of these compounds and of the liposomes,produced therewith is, however, limited to in vitro or ex vitro uses,since such positively charged liposomes form uncontrolled aggregateswith serum components.

[0008] The limitation to very few pK values, generally to that of thecarboxyl group in the cholesterol hemisuccinate (approximately 4.5) is adisadvantage of the pH-sensitive liposomes, which are availableaccording to the state of the art. A further disadvantage of thecompounds is the limitation to negative charge carriers. These are notsuitable for binding nucleic acids and, frequently also, proteinsefficiently.

[0009] Cationic liposomes show good bonding of nucleic acids andproteins and are in a position to bring these active ingredients intocells. It is a disadvantage that they cannot be used for in vivoapplications.

[0010] It was therefore an objective to produce the liposomalstructures, which

[0011] i) permit an efficient inclusion of active in agents,

[0012] ii) can transport these active ingredients into biological cells,

[0013] iii) are compatible with use under in vivo conditions and

[0014] iv) can be produced simply and inexpensively.

[0015] The inventive object is accomplished by amphoteric liposomes,which comprise at least one positive and at least one negative chargecarrier, which differs from the positive one, the isoelectric point ofthe liposomes being between 4 and 8. This objective is accomplishedowing to the fact that liposomes are prepared with a pH-dependent,changing charge.

[0016] Liposomal structures with the desired properties are formed, forexample, when the amount of membrane-forming or membrane-based cationiccharge carriers exceeds that of the anionic charge carriers at a low pHand the ratio is reversed at a higher pH. This is always the case whenthe ionizable components have a pKa value between 4 and 9. As the pH ofthe medium drops, all cationic charge carriers are charged more and allanionic charge carriers lose their charge.

[0017] The following abbreviations are used in connection with theinvention: CHEMS cholesterol hemisuccinate PC phosphatidyl choline PEphosphatidyl ethanolamine PS phosphatidyl serine PG phosphatidylglycerol Hist-Chol histidinyl cholesterol hemisuccinate

[0018] The membrane-forming or membrane-based charge carriers have thefollowing general structure of an amphiphile:

[0019] charge group-membrane anchor

[0020] The naturally known systems or their technically modified formscome into consideration as membrane anchors. These include, inparticular, the diacyl glycerols, diacyl phosphoglycerols(phospholipids) and sterols, but also the dialkyl glycerols, thedialkyl- or diacyl -1-amino-2,3-dihydroxypropanes, long-chain alkyls oracyls with 8 to 25 carbon atoms, sphingolipids, ceramides, etc. Thesemembrane anchors are known in the art. The charge groups, which combinewith the these anchors, can be divided into the following 6 groups:

[0021] Strongly cationic, pKa>9, net positive charge: on the basis oftheir chemical nature, these are, for example, ammonium, amidinium,guanidium or pyridinium groups or timely, secondary or tertiary aminofunctions.

[0022] Weakly cationic, pKa<9, net positive charge: on the basis oftheir chemical nature, these are, in particular, nitrogen bases such aspiperazines, imidazoles and morpholines, purines or pyrimidines. Suchmolecular fragments, which occur in biological systems, preferably are,for example, 4-imidazoles (histamine), 2-, 6-, or 9-purines (adenines,guanines, adenosines or guanosines), 1-, 2-or 4-pyrimidines (uraciles,thymines, cytosines, uridines, thymidines, cytidines) or alsopyridine-3-carboxylic acids (nicotinic esters or amides).

[0023] Nitrogen bases with preferred pKa values are also formed bysubstituting nitrogen atoms one or more times with low molecular weightalkane hydroxyls, such as hydroxymethyl or hydroxyethyl groups. Forexample, aminodihydroxypropanes, triethanolamines,tris-(hydroxymethyl)methylamines, bis-(hydroxymethyl)methylamines,tris-(hydroxyethyl)methylanines, bis-(hydroxyethyl)methylarnines or thecorresponding substituted ethylamines.

[0024] Neutral or zwitterionic, at a pH from 4 to 9: on the basis oftheir chemical nature, these are neutral groups, such as hydroxyls,amides, thiols or zwitterinonic groups of a strongly cationic and astrongly anionic group, such as phosphocholine or aminocarboxylic acids,aminosulfonic acids, betaines or other structures.

[0025] Weakly anionic, pKa>4, net negative charge: on the basis of theirchemical nature, these are, in particular, the carboxylic acids. Theseinclude the aliphatic, linear or branched mono-, di- or tricarboxylicacids with up to 12 carbon atoms and 0, 1 or 2 ethylenically unsaturatedbonds. Carboxylic acids of suitable behavior are also found assubstitutes of aromatic systems.

[0026] Other anionic groups are hydroxyls or thiols, which candissociate and occur in ascorbic acid, N-substituted alloxane,N-substituted barbituric acid, veronal, phenol or as a thiol group.

[0027] Strongly cationic, pKa<4, net negative charge: on the basis oftheir chemical nature, these are functional groups such as sulfonate orphosphate esters.

[0028] Amphoteric charge carriers, pI between 4.5 and 8.5, net positivecharge below the pI, net negative charge above the pI: on the basis oftheir chemical nature, these charge carriers are composed of two or morefragments of the groups named above. For carrying out the invention, itis, initially, immaterial whether the charged groups are on one and thesame membrane anchor or if these groups are on different anchors.Amphoteric charge carriers with a pI between 5 and 7 are particularlypreferred for implementing the invention.

[0029] Strongly cationic compounds are, for example:

[0030] DC-Chol 3-β-[N-(N′,N′-dimethylmethane) carbamoyl] cholesterol,

[0031] TC-Chol 3-β-[N-(N′, N′, N′-trimethylaminoethane) carbamoylcholesterol

[0032] BGSC bisguanidinium-spermidine-cholesterol

[0033] BGTC bis-guadinium-tren-cholesterol,

[0034] DOTAP (1,2-dioleoyloxypropyl)-N,N,N-trimethylammonium chloride

[0035] DOSPER (1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylarnide

[0036] DOTMA (1,2-dioleoyloxypropyl)-N,N,N-trimethylamronium chloride)(Lipofectin®)

[0037] DORIE (1,2-dioleoyloxypropyl)-3-dimethylhydroxyethylammoniumbromide

[0038] DOSC (1,2-dioleoyl-3-succinyl-sn-glyceryl choline ester)

[0039] DOGSDSO (1,2-dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyldisulfide omithine)

[0040] DDAB dimethyldioctadecylammonium bromide

[0041] DOGS ((C18)₂GlySper3⁺) N,N-dioctadecylamido-glycol-spermin(Transfectam®) (C18)₂Gly⁺ N,N-dioctadecylamido-glycine

[0042] CTAB cetyltrimethylarnmonium bromide

[0043] CpyC cetylpyridinium chloride

[0044] DOEPC 1,2-dioleoly-sn-glycero-3-ethylphosphocholine or otherO-alkyl-phosphatidylcholine or ethanolamines,

[0045] amides from lysine, arginine or omithine and phosphatidylethanolarnine.

[0046] Examples of weakly anionic compounds are: His-Cholhistarninyl-cholesterol hemisuccinate, Mo-Cholmorpholine-N-ethylamino-cholesterol hemisuccinate or histidinyl-PE.

[0047] Examples of neutral compounds are: cholesterol, ceramides,phosphatidyl cholines, phosphatidyl ethanolamines, tetraether lipids ordiacyl glycerols.

[0048] Examples of weakly anionic compounds are: CHEMS cholesterolhemisuccinate, alkyl carboxylic acids with 8 to 25 carbon atoms ordiacyl glycerol hemisuccinate. Additional weakly anionic compounds arethe amides of aspartic acid, or glutarnic acid and PE as well as PS andits amides with glycine, alanine, glutamine, asparagine, serine,cysteine, threonine, tyrosine, glutamic acid, aspartic acid or otheramino acids or aminodicarboxylic acids. According to the same principle,the esters of hydroxycarboxylic acids or hydroxydicarboxylic acids andPS are also weakly anionic compounds.

[0049] Strongly anionic compounds are, for example: SDS sodium dodecylsulfate, cholesterol sulfate, cholesterol phosphate, cholesterylphosphocholine, phosphatidyl glycerols, phosphatid acids, phosphatidylinositols, diacyl glycerol phosphates, diacyl glycerol sulfates, cetylphosphate or lyosophospholipids.

[0050] Amphoteric compounds are, for example,

[0051] Hist-Chol Nα-histidinyl-cholesterol hemisuccinate,

[0052] EDTA-Chol cholesterol ester of ethylenediamine tetraacetic acid

[0053] Hist-PS Nα-histidinyl-phosphatidylserine or N-alkylcarnosine.

[0054] The inventive liposomes contain variable amounts of suchmembrane-forming or membrane-based amphiphilic materials, so that theyhave an amphoteric character. This means that the liposomes can changethe sign of the charge completely. The amount of charge carrier of aliposome, present at a given pH of the medium, can be calculated usingthe following formula:

z=Σni((qi−1)+10^((pK−pH))/(1+10^((pK−pH)))

[0055] in which

[0056] qi is the absolute charge of the individual ionic groups belowtheir pK (for example, carboxyl=0, simple nitrogen base=1, phosphategroup of the second dissociation step =−1, etc.)

[0057] ni is the number of these groups in the liposome.

[0058] At the isoelectric point, the net charge of the liposome is 0.Structures with a largely selectable isoelectric point can be producedby mixing anionic and cationic portions.

[0059] The structures can also be constructed so that, in particular, asthe pH drops, the charge on the molecule as a whole is actually changedfrom negative to positive. Such a reversal of charge is advantageousparticularly when the liposomes, produced with these structures, are tobe used in physiological interrelationships. Only liposomes with anoverall negative charge are compatible with blood and serum components.A positive charge leads to aggregations. Liposomes with a positivecharge are, however, very good fusogenically and can transport activeingredients into cells. A pH-dependent reversal of charge thereforepermits compounds to be constructed, which are compatible with serumbecause they have a negative charge; however, after their endocytoticabsorption, their charge is reversed and they become fasogenic only inthe cell.

[0060] In a preferred embodiment of an embodiment of the invention, theamphoteric liposomes have an isoelectric point between 5 and 7.

[0061] The invention also relates to amphoteric liposomes, whichcomprise at least one amphoteric charge carrier, the amphoteric chargecarrier having an isoelectric point of between 4 and 8.

[0062] In a preferred variation, the amphoteric charge carrier of theliposomes has an isoelectric point of between 5 and 7.

[0063] The invention also relates to amphoteric liposomes, the liposomescomprising at least one amphoteric charge carrier and an anionic and/orcationic charge carrier.

[0064] It is appropriate that, in a preferred variation, the amphotericliposomes have an isoelectric point between 5 and 7.

[0065] In a special variation of the invention, the inventive liposomescomprise phosphatidyl choline, phosphatidyl ethanolamine, diacylglycerol, cholesterol, tetraether lipid, ceramide, sphigolipid, and/ordiacyl glycerol. However, the preparation of the liposomes can, ofcourse, be carried out with many lipid combinations of the inventiveteachings. For examples, liposomes can be synthesized using a largeamount of CHEMS (about 40%) and a smaller amount of DOTAP (about 30%).At the pK of the carboxyl group of the CHEMS, the negative charge ofthis component is already suppressed so far, that the positive chargecarrier predominates overall. An alternative formulation is the mixingof CHEMS with HisChol the stronger charging of the positive chargecarrier HisChol going along synergistically with the discharging of thenegative CHEMS.

[0066] If Hist-Chol, which in itself is amphoteric, is incorporated in aneutral membrane of, for example, phosphatidyl choline, an amphotericliposome with an isoelectric point, which largely corresponds to that ofHist-Chol, also results.

[0067] It is known to those skilled in the art how the importantparameters can be adapted by manifold variations of the inventiveteachings:

[0068] (i) the charge density of the liposomes at the end points of theof the charge reversals by the amount and the pKa values of the chargecarriers used,

[0069] (ii) the slope of the charge reversal curve by the ratio of thetwo charge carriers, by their absolute amounts and by an optimallysynergistic effect of two complementary pH-sensitive lipids and

[0070] (iii) the passing of the zeta potential through zero due to theratio of the two charge carriers or also due to the position of the pKvalue or values.

[0071] In a further variation of the invention, the liposomes have anaverage size of between 50 and 1000 nm, preferably of between 70 and 250nm and particularly between 60 and 130 nm. The amphoteric liposomes aresynthesized by methods known in the art, such as the injection ofethanol into a lipid solution in an aqueous buffer, by hydrating drylipid films or by detergent dialysis. The size of the liposomes canvary, generally between 50 nm and 10,000 nm. Homogeneous populations canbe produced by high-pressure homogenization or by extrusion.

[0072] In a preferred variation of the invention, the liposomes comprisean active ingredient.

[0073] Advisably, in a preferred variation, the active ingredient is aprotein, a peptide, a DNA, an RNA, an antisense nucleotide and/or adecoy nucleotide.

[0074] In a farther preferred variation of the invention, at least 80%of the active ingredient in the interior of the liposome.

[0075] The invention also relates to a method for charging a liposomewith active ingredient, a defined pH being used for the encapsulationand the pH being adjusted to a second value for separating the unboundmaterial.

[0076] The invention furthermore also relates to a method for charging aliposome with active ingredient, the liposomes being permeabilized andclosed at a defined pH.

[0077] The invention also relates to the use of the liposomes for thepreparation of nanocapsules by depositing polymers or polyelectrolyteson the lipid layer. Such substances can be precipitated once or severaltimes on the surface. With a repeated deposition, which optionally canbe carried out in the absence of cross-linking agents, liposomalnanocapsules of the type described in the WO 00/28972 or in theW001/64330 are formed. It is advantageous that the electrostaticinteraction with the polyelectrolyte can be interrupted when thesubstances described here are used. It is known that the interaction ofa polyelectrolyte with charge carriers of the liposomal membrane canlead to the de-mixing of membrane components and to the formation oflipid clusters. In many cases, this de-mixing is associated with apermeabilization of the liposome. The inventive substances enable thisinteraction to be switched off after the coating process. The liposomesare enclosed only sterically in the nanocapsules if the pH is increasedat this time and there no longer is any interaction between the membraneand the polyelectrolyte. Cluster formation of the lipids and thepermeabilization of the membrane, associated therewith, can thus beavoided.

[0078] The invention also relates to the use of the inventive liposomesfor packaging and releasing active ingredients. In this variation, theliposomes bring about, in particular, the efficient packaging of activeingredients, such as nucleic acids. Nucleic acids are incubated withsaid lipids particularly at a low pH (about 3 to 6). After the formationof the liposomes, nucleic acids, adhering to the outside, can be washedoff by changing to a high pH (about 7 to 9).

[0079] An analogous procedure can be used to package proteins.Advantageously, the pH of the medium is adjusted to a value here, whichlies between the pI of the liposome and that of the protein. It hasproven to be particularly advantageous, if the two pI values are morethan one unit apart.

[0080] In a farther variation of the invention, the liposomes are usedto prepare release systems in diagnostics.

[0081] In a further preferred variation of the invention, the liposomesare used as transfection systems, that is, for bringing activeingredients into cells.

[0082] In a further variation of the invention, the liposomes are usedfor the controlled release of their contents by fusion orpermeabilization of the membrane. For example, liposomes of a lipid,which by itself is not membrane-forming, can be stabilized by theincorporation of charge carriers, such as PE. If the charge carrier istransformed into a neutral, uncharged or zwitterionic state, thepermeability of the membrane is increased. Known liposomes of the stateof the art (PE/CHEMS, Tachibana et al.) permit such a permeabilizationat the low pH values, which are attained under physiological conditionsonly in the interior of endosomes or during passage through the stomach.Amphoteric liposomes can be produced by the measures listed above insuch a manner, that their neutral point lies at any desirable pH between4 and 9. Under these conditions, the liposomes are permeable and candeliver cargo to the medium.

[0083] However, the liposomal formulations can be produced, processedand stored under conditions of lesser permeability. In a preferredembodiment of the invention, liposomes are produced so that they releaseof their cargo under conditions of a physiological pH, but enclose theircargo securely at a low pH. Such liposomes are suitable particularly forthe preparation of formulations with slow release kinetics, the releasebeing initiated only by contact with body fluids and not during storageor transport.

[0084] A preferred embodiment of the inventive teaching thereforeconsists of the use of such liposomes for therapeutic purposes,especially for such uses, which employ the specific targeting of theliposomes. The slight nonspecific binding is a prerequisite here fortransporting the liposomes to the target place. In contrast to this, ahigh nonspecific binding would prevent any transport of the liposomes tothe target place. A specific binding can be attained by further measuresof the state of the art, that is, by selecting the size of the liposomesor also by binding the ligands to the liposomal surface, which binds toa target receptor of the cell surface. Ligands may, for example, beantibodies or their fragments, sugars, hormones, vitamins, peptides,such as arg-gly-asp (RGD), growth factors, bilirubin or othercomponents.

[0085] The preferred variation of the inventive teachings relates to theuse of the liposomes for therapeutic or diagnostic applications under invivo conditions. Preferably, such liposomes are ones, which have aslight nonspecific binding and, with that, a slight tendency to fuseunder physiological conditions, but are combined strongly and with ahigh fusion competence under changed conditions. Such liposomes areamphoteric liposomes, which have an overall anionic particle chargeunder physiological conditions and an increasingly cationic charge at apH below 6.5. Such pH values occur during the endocytosis of theliposomes into cells. Such pH values also occur in the interior oftumors and in the external layers of the skin. Low pH values can also beobtained by perfusing an organ ex vivo for a certain period of time. Ahigh binding strength and fusion competence is therefore limited tothose liposomes, which were already taken up by cells or special tissue.The binding strength and increasing fusion competence support the fusionof the liposomal membrane with the cell membrane. This event leads to adirect release of the cargo into the interior of the cell withoutreleasing components of the lysis of the endosome and, with that,endangering the cargo or cell components.

[0086] Furthermore, the use of the liposomes as a sustained releaseformulation and/or as a circulating depot is appropriate. The liposomescan also be used advantageously for intravenous or peritonealapplication. In a particularly preferred variation of the invention, theliposomes are used as a vector for the in vivo, in vitro and ex vivotransfection of cells.

[0087] The inventive liposomes have several advantages. Cationicallychargeable liposomes of 40 percent HisChol and PC bind the nucleicacids, such as DNA, to their membrane even under conditions of a neutralpH. Surprisingly, this binding is suppressed completely if theabove-mentioned liposomes are produced using 5 percent of PG in additionand then have amphoteric properties. However, the binding of nucleicacids to the membrane can be restored once again by decreasing the pH.The inventive liposomes are therefore particularly well suited for thepH-dependent binding of nucleic acids.

[0088] Furthermore, it was surprisingly found that a series of proteinsalso behaves in the manner described for nucleic acids. For example,antibodies bind not at a neutral pH, but under slightly acidicconditions effectively to the membrane of the inventive liposomes. Sucha behavior cannot be observed in the case of pH-sensitive liposomes froma neutral lipid and CHEMS nor from such a liposomes from a neutral lipidand HisChol. It is therefore a special property of the amphotericliposomes. Surprisingly, it was also found that inventive liposomes,contrary to the known, constitutive, cationic liposomes, are compatiblewith serum. An appropriate embodiment of the inventive teachingstherefore consists of the use of such liposomes for therapeuticproperties. It is an advantage of the liposomes that, in comparison toknown, constitutive, cationic liposomes, the nonspecific binding tocells is significantly less.

[0089] It is, however, also surprising that the fusion competence of theinventive liposomes depends on the pH of the medium. In comparison tobiological membranes of cells, the fusion competence is determined bythe lipid selected and also by the charging of the liposomes. Usually, abinding step precedes the actual fusion. However, strong binding of theliposomes to cell membranes is not always desirable and should takeplace, as described above, only under controlled conditions inparticular cells or tissue.

[0090] The liposomes can therefore by used to construct liposomalvectors for the transport of active ingredients into cells. Allmaterials, which do not form micelles, come into consideration as activeingredients. Water-soluble materials are particularly suitable as activeingredients. They include many proteins and peptides, especiallyantibodies or enzymes or antigens, all nucleic acids, independently oftheir molecular weight and their derivation from RNA or DNA. However,they include also other biological macromolecules, such as complexsugars, natural products and other compounds, as well as low molecularweight active ingredients of synthetic or natural origin, whichotherwise cannot penetrate through the cell membrane as barrier. Withthe help of vectors, such materials can then be transported into theinterior of cells and initiate actions, which are not possible withoutthis transport.

[0091] Accordingly, with the help of the inventive teachings, liposomescan be prepared, the fusion and binding properties of which differ atdifferent pH values. Serum-compatible liposomes, which are laden with alarge amount of active ingredients and transport these into the interiorof cells, can therefore be produced in this way. Someone, skilled in theart, is able to combine elements of the inventive teachings with oneanother and, with that, produce liposomes, which are optimally suitablefor a particular purpose.

[0092] The invention is described in greater detail in the following bymeans of examples without being limited to these examples.

EXAMPLE 1 Preparation And Charge Properties Of Amphoteric Liposomes WithCharge Carriers, Which Can Be Charged Positively And Are ConstantlyCharged Negatively

[0093] His-Chol (5 mg) and 7.8 mg of POPC and 2 mg of DPPG are dissolvedin 4 ml of a 1:1 (v/v) mixture of chloroform and methanol and driedcompletely in a rotary evaporator. The lipid film is hydrated with 4.3mL of the appropriate buffer (10 mM KAc, 10 mM HEPES, 150 mM NaCl, pH7.5, in a lipid concentration of 5 mM by a five-minute treatment withultrasound. Subsequently, the suspension is frozen and, after thawing,extruded several times (Avestin LiposoFast, polycarbonate filter with a200 nm pore width). For measuring the zeta potential, the finalconcentration of the liposomes is adjusted to a value of 0.2 mM. For thedilution, the buffer system, named above, is used at a pH of 7.5 or 4.2.The zeta potentials measured lie between −18 mV (at pH 7.5) and +35 mV(at pH 4.2).

EXAMPLE 2 Preparation And Charge Properties Of Amphoteric Liposomes WithConstant Positive And Variable Negative Charge Carriers

[0094] POPC, DOTAP and CHEMS are dissolved in the molar ratios givenbelow in 4 mL of a 1:1 (v/v) mixture of chloroform and methanol andevaporated completely in the rotary evaporator. The lipid film ishydrated with 4.3 mL of the appropriate buffer (10 mM KAc, 10 mM HEPES,150 mM NaCl, pH 7.5, in a total lipid concentration of 5 mM by afive-minute treatment with ultrasound. Subsequently, the suspension isfrozen and, after thawing, excluded repeatedly (Avestin LiposoFast,polycarbonate filter with a 200 nm pore width). The Table below showsthe zeta potentials as a function of pH.

[0095] Composition of the liposomes in mole percent liposome 1 POPC 50DOTAP 40 Chems 10 liposome 2 POPC 50 DOTAP 30 Chems 20 liposome 3 POPC50 DOTAP 25 Chems 25 liposome 4 POPC 50 DOTAP 20 Chems 30 liposome 5POPC 50 DOTAP 40 Chems 10

[0096] TABLE 1 Zeta Potentials in mV Liposome Liposome Liposome LiposomeLiposome pH 1 2 3 4 5 4 44.2 38.4 34.7 31.7 16.2 5 39.9 25.6 27.2 22.13.3 6 37 21.4 16.4 2.5 −7.3 7.5 29.2 1.8 −7.9 −18.9 −34.6

[0097] The height of the zeta potential and its slope can be selectedwithin why limits by means of a suitable composition.

EXAMPLE 3 Preparation And Charge Properties Of Amphoteric Liposomes WithComplete Switchability In One Compound

[0098] His-Chol (5 mg) and 9.8 mg of POPC are dissolved in 4 ml of a 1:1(v/v) mixture of chloroform and methanol and dried completely in arotary evaporator. The lipid film is hydrated with 4.3 niL of theappropriate buffer (10 mM KAc, 10 mM HEPES, 150 mM NaCl, pH 7.5, in alipid concentration of 5 mM by a five-minute treatment with ultrasound.Subsequently, the suspension is frozen and, after thawing, extrudedseveral times (Avestin LiposoFast, polycarbonate filter with a 200 nmpore width). The course of the zeta potential at different pH values andionic strengths is shown in the table below (Table 2). TABLE 2 pHWithout Salt 100 mM of NaCl 4 45.6 20.2 5 26.9 2.2 6 −4.1 −5.2 7 −31.4−15.3 8 −45.7 −25.4

EXAMPLE 4 Serum Aggregation

[0099] Lipid films are prepared as in Example 1. A lipid mixture, whichdid not contain any DPPG, was used as comparison sample. The lipid filmswere hydrated in buffer (10 mM of phosphate, 150 mM of sodium chloride,pH of 7.4) and extruded as above. Human serum is diluted with an equalamount of buffer (10 nmm of phosphate, 150 mM of sodium chloride, pH of7.4), particular components and fat being removed by centrifuging (20minutes, 13,000 rpm, 4° C.); the clear serum is filtered sterile with afilter having a pore width of 0.2 μm.

[0100] The liposomes, prepared above are added to the serum inconcentration of 1 mM and incubated for 15 minutes at 37° C. After theincubation, the suspension of the DPPG-containing liposomes is uniformlycloudy; however, flocculation cannot be observed. The diameter of theliposomes is deternined by means of dynamic light scattering and ischanged by less than 10% from that of the starting sample. Thesuspension of the DPPG-free liposomes clearly shows flocculation.

EXAMPLE 5 Stability of the Membrane

[0101] Aside from serum aggregation, the precipitation of an activeingredient (carboxyfluorescein, CF) in the presence of human serum wasalso investigated. For this purpose, POPC/DOTAP/CHEMS liposomes ofdifferent decomposition were prepared by the method of Example 2: POPC100% (as control), POPC/DOTAP/CHEMS 60:30:10, 60:20:20 and 60:10:30 (inmole %). Any CF, which is not enclosed, was removed by gel filtration.For the measurement, the liposomes were diluted to 0.1 mM in serum andincubated at 37° C. A 30 μL sample was removed at certain times anddiluted to 300 μL with 100 M of tris buffer, having a pH of 8.2 and thefluorescence was measured. The 100% values were obtained by dissolvingthe liposomes with 10 μL of Tritron X-100 (10% in water). The enclosedCF as a finction of time is shown in the Table below.

[0102] The liposomes lose only a little CF into the serum during the4-hour period of rement. POPC/DOTAP/CHEMS 60:30:10 and 60:20:20 stillcontain about 75%, and POPC/DOTAP/CHEMS 60:10:30 even 100% of theiroriginal CF content (see 3). TABLE 3 Time in POPC/DOTAP/CHEMSPOPC/DOTAP/CHEMS POPC/DOTAP/CHEMS Min. POPC 60:30:10 60:20:10 60:10:30 0100%  100%  100%  100% 15 91% 84% 95% 107% 60 94% 81% 87% 110% 120 96%80% 76% 105% 240 96% 80% 77% 107%

EXAMPLE 6 Binding DNA

[0103] Liposomes of the following compositions (in mole %) are preparedas in Example 1 (all data is in mole %). A: 60 POPC 40 HisChol B: 55POPC 40 HisChol 5 CHEMS C: 60 POPC 20 HisChol 20 CHEMS

[0104] The liposomes are suspended in a concentration of 0.2 mM inbuffer (10 mnM of potassium acetate, 10 mM of HEPES, pH 4.2 or 7.5). ADNA solution (45 μL, 1 mg of DNA (Hering sperm, SIGMA D3159) in 1 mL ofwater) are added in each case to 1 mL of the different liposomes samplesand mixed quickly. After an incubation period of 15 minutes, the sampleis filled up with 6 mL of the appropriate buffer and the zeta potentialof the liposomes is measured (Table 4). TABLE 4 pH 4.2 pH 7.5 Lipid −DNA+DNA −DNA +DNA A +47.6 −32.0 +2.4 −44.4 B +47.8 −28.1 +0.1 −38.4 C +34.0−28.6 −10.1 −24.7

[0105] Under the conditions of an excess of cationic charges (pH 4:2),there is a strong reversal of the charge of the particles. At a neutralpH of 7.5, the CHEMS in high concentration (liposome C) canovercompensate the charge of the HisChol and the particles have anegative zeta potential. Only slight amounts of DNA bind to suchparticles.

EXAMPLE 7 Binding and Detaching DNA

[0106] Liposomes having the compositions POPC/DOTAP/CHEMS in the ratioof 60:15:25 and POPC/DCChol/CHEMS in the ratio of 60:15:25 (in mole %),were prepared by the method of Example 2. The binding of the DNA wascarried out at a pH of 4.2 by the method of the above example and thezeta potentials were determined. Subsequently, the pH of the samples wasadjusted to a value of 7.5 and the zeta potential was measured onceagain. Mixture Zeta (mV) a) POPC/DCChol/CHEMS 60:15:25 (pH 4.2) −43.5(aggregate) b) POPC/DOTAP/CHEMS −43.7 c) POPC/DCChol/CHEMS −18.5 d)POPC/DOTAP/CHEMS −14.5

[0107] In the presence of DNA, a negative zeta potential is measured ata low pH; however, the original particles were charged positively. Afterthe change to the neutral pH, this charge, which is due to the DNA, isdecreased. The zeta potentials approach that of the untreated liposomes(−11 mV at a pH of 7.5).

EXAMPLE 8 DNA Inclusion and Detachment of Material not Encapsulated

[0108] Two liposome formulations, having compositions ofPOPC60/DOTAP15/CHEMS25 and POPC85/DOTAP15 respectively, are prepared asdry lipid films as described above. In each case, the total amount oflipid was 4 μmoles. For hydration, Herings DNA was dissolved in 10 mM ofpotassium acetate, 10 mM of HEPES and 100 mM of sodium chloride at a pHof 4.0. The DNA (4 mg) was added directly to the lipid films. Theresulting liposomes were frozen and thawed repeatedly and subsequentlyextruded through a 200 nm filter.

[0109] Each 500 μL of particles was mixed with 2.5 mL of a sucrosesolution (0.8M sucrose in the above buffer, at a pH of 4.0 or 7.5). Overthis, 1.5 mL of a 0.5 M sucrose solution and 0.5 mL of the buffer wereplaced.

[0110] Liposomes were then separated by flotation from unbound DNA.After the flotation, the liposomes were removed from the buffer/0.5 Msucrose interface. The amount of bound DNA was determined byintercalation of propidium iodide. The Stewart assay was used todetermine the amount of lipid. Only the PC used responds in the Stewartassay. The other lipids were not calculated by means of this value. Theresults are shown in the Table below (Table 5). TABLE 5 Liposome pH 4.0pH 7.5 POPC/DOTA/ 2 μg DNA/μg DOTAP 1.2 μg DNA/μg DOTAP CHEMS 60/15/25POPC/DOTAP 2.3 μg DNA/μg DOTAP 2.3 μg DNA/μg DOTAP 85/15

[0111] With the amphoteric liposomes, only about half of the bound DNAfloats up after the change in pH to 7.5. This material is the actuallyenclosed material. Similar results are obtained by digesting with DNAse

[0112] DNA cannot be detached once again from constitutively cationicliposomes by changing the pH or by additionally increasing the ionicstrength and always remains on the outside.

EXAMPLE 9 Fusion Properties

[0113] Liposomes with the following compositions are prepared as inExample 1 (all data in mole %): A) POPC 60 HisChol 40 B) POPC 55 HisChol40 CHEMS 5 X) POPC 100 Y) POPC 60 DPPG 40

[0114] The facultative cationic liposomes A or B are incubated with theneutral liposomes X or the anionic liposomes Y in the buffer (10 mMHEPES, 10 mM potassium acetate, pH 4.2 or 7.5). The possible fusion ofliposomes is analyzed by size measurement by means of dynamic lightscattering (Table 6). TABLE 6 Liposome 1 X X Y Y Liposome 2 A B A B pH4.2 161.6 nm 191.9 nm 1689.3 nm 2373.2 nm pH 7.5 191.8 nm 202.4 nm 250.0 nm  206.3 nm

[0115] The starting sizes of the liposomes were 161.8 nm at pH 4.2 and165.9 nm at pH7.5

[0116] A) 183.2 nm

[0117] X) 195.2 nm

[0118] Y) 183.2 nm

[0119] The size of the pairs with the complementary charge (YA and YB)differs clearly from the size of the mixed suspensions with the neutralliposome X. The extent of the interaction is determined by the magnitudeof the charge of the facultative cationic liposomes. The extent of thefusion to larger units does not depend on the fusogenic lipid PE.

EXAMPLE 10 Permeability to Macromolecules

[0120] DOPE (13.75 μmoles), 2.5 μmoles of CHEMS and 10 μmoles of HisCholare dissolved in isopropanol and the solvent is drawn off under avacuum. A solution (2.5 ML) of proteinase K in buffer (1 mg/mL ofproteinase K, 10 mM of potassium acetate, 10 mM HEPES, 150 mM of sodiumchloride, pH 4.2) is added to the dried lipid film. After the film ishydrated, the liposomes formed are extruded through a 400 nm membrane.Proteinase, which is not enclosed, is removed by floatation of theliposome in the sucrose gradient. The liposomes, so produced, areincubated with 7.5 ML of buffer at a pH of 4.2 and 7.2 (buffer as above,starting pH 4.2 and 8.0). After the combination, the proteinase Kreleased is removed using a 0.1 μm membrane. The liposomes, remaining inthe filter, are then treated with 7.5 mL of a solution of Triton X-100in buffer (as above, pH 8.0).

[0121] All filtrates are tested for the presence of proteinase K. Forthis purpose, a solution of azocasein (6 mg/mL of azocasein in 1 M urea,200 mM tris sulfate, pH 8.5) is used. This solution (500 μL) is mixedwith 100 μL of filtrate or buffer and incubated for 30 minutes at 37° C.The reaction is terminated by the addition of 10% trichloroacetic acid.Precipitated proteins are removed by centrifuging. The coloration ismeasured at 390 nm (Table 7). TABLE 7 Absorption at 390 nm pH ofIncubation Triton X-100 Blank 4.2 − 0.0192 4.2 + 0.2345 7.2 − 0.22107.2 + 0.0307

[0122] If the incubation of the liposomes is carried out at a pH ofabout 4.2, very little if any proteinase K is released. Only thedissolution of the liposomes with Triton X-100 leads to the release ofthe enzyme. If the liposomes are incubated at a pH of 7.2, the bulk ofthe enzyme is released already without the addition of the Triton and isfound in the first filtrate. Hardly any additional enzyme is thendissolved from the liposomes by the addition of Triton.

EXAMPLE 11 Protein Binding

[0123] Liposomes, having the composition POPC50/DOTAP10/CHEMS40 (alldata in mole %) are prepared as in the preceding examples. A solution of0.26 mg/mL of lysozyme in buffer (10 mM MES of pH 5.0 or pH 6.0 or 10 mMof HEPES of pH 7.0 or pH 8.0) is used to hydrate the lipid film. Afterthe hydration, all samples were frozen and thawed repeatedly.Subsequently the liposomes are homogenized by ultrasound and extrudedthrough a 200 nm filter.

[0124] The liposome suspension, so prepared, is adjusted to a pH of 4.0by the addition of acetic acid. Subsequently the liposomes are separatedby flotation from protein, which has not been incorporated. Theproportion of enclosed protein is given in the Table below (Table 8).TABLE 8 pH during Inclusion % of Material Enclosed 5.0 4 6.0 21 7.0 758.0 80

[0125] Liposomes of the composition used show a pI of 5; the lysozyme isa basic protein with a pI of 11.5. The two partners therefore haveopposite charges at a pH between 6 and 8. An efficient inclusion in theliposomes is brought about by electrostatic attraction. Protein, notencapsulated, was removed at a pH of 4. The interaction between thepartners is cancelled at this pH.

EXAMPLE 12 Transfection Into Cells

[0126] HeLa cells or CHO cells (3×10⁵) were plated into each cavity of a6-well titer plate and cultured for three days. Liposomes(POPC/DOTAP/CHEMS 60/30/10) were prepared in the presence offluorescence-labeled dextran (TRITC dextran 10 mg/mL in the hydrationbuffer). TRITC dextran, which had not been incorporated, was removed bygel filtration. The liposomes, so prepared, were added to the cells andincubated for 6 hours at 37° C. Subsequently, the cells were washedtwice with buffer. The absorption of dextran was followed in themicroscopic image. The results are shown in FIG. 1.

EXAMPLE 13 Ligand Binding and Transfection

[0127] Liposomes, having the composition ofPOPC/DOTAP/Chems/N-glutaryl-DPPE (50:10:30:10 (mole %)) are prepared asin Example 2. At the same time, they are hydrated with a solution of 3mg/mL of TRITC-Dextran (having a molecular weight of about 4,400) inHEPES 10 mM and 150 mM of sodium chloride at a pH of 7.5. TRITC-Dextran,which is not enclosed, is removed by gel filtration through a SephadexG-75 column. Activation of the N-glutaryl DEPPs with EDC(1-ethyl-3-(3-dimethylaminopropyl carbodiimide) (3.5 mg of EDC per 400μL of liposome suspension) and subsequent stirring in the dark for 5hours brings about the binding of the cyclic peptide RCDCRGDCFC to theliposomal surface. The RGD peptide (250 μg in 150 μL of buffer) was thenadded and stirring was continued overnight. The liposomes were separatedby gel filtration from the peptide, which had not been bound.

[0128] Human endothelium cells (HUVEC) were cultured in a specialmedium. The liposomes, modified with ligand, and control liposomeswithout RGD ligand were added as a 0.5 mM suspension to the cells. After2 hours, the liposomes are removed and the cell chambers rinsed 3 timeswith PBS buffer and viewed under the fluorescence microscope. The TRITCfluorescence of cells, which had been treated with RDG liposomes, isdistinctly more red than that of the control liposomes.

EXAMPLE 14 Pharmacokinetics (Blood Level and Organ Distribution ofpH-Switchable Liposomes)

[0129] Liposomes of POPC/Chol (60:40), POPC/Hist-Chol/Chol (60:20:20)and POPC/DOTAP/Chems (60:10:30) (500 μL) were injected into the tailvein of male Wistar rats.

[0130] Liposome suspensions (50 mM) were prepared by hydrating a lipidfilm of the corresponding formulation (addition of 0.03 moles of[14]C-DPPC) with 2 mL of a solution of 1 mg [3]H-inulin in HEPES 10 mM,sodium chloride 150 nm at a pH of 7.5). After 3 freezing and thawingcycles, the suspensions were extruded repeatedly through a 400 nmmembrane (LiposoFast, Avestin). [3]H-Inulin which had not been enclosed,was removed by gel filtration though a G-75 Sephadex-column andsubsequent concentration over CENTRIPREP (Millipore) centrifuging units.

[0131] Liposome suspension (0.5 mL) was administered to 4 experimentalanimals per formulation and blood samples were taken after 5 minutes, 15minutes, 60 minutes, 3 hours, 12 hours and 24 hours. The radioactivityof the membrane fraction and of the soluble cargo was measured byscintillation and gave the following values:

[0132] Elimination half-life times from the blood POPC/Chol greater than120 minutes POPC/DOTAP/Chems greater than 120 minutes POPC/Hist-Cholgreater than 120 minutes

[0133] With their relatively long half-life in the blood, the inventiveliposomes fulfill the basic prerequisites for a vector system. They arenot acutely toxic and not absorbed immediately by theirreticuloendothelial system. Up to the end of the experiment, the ratioof the 3[H] to the 14[C] radioactivity of the blood samples wasconstant. Release of the cargo by complement lysis therefore does nottake place in any of the cases.

1. Amphoteric liposomes, wherein the liposomes comprise at least onepositive charge carrier and at least one negative charge carrier, whichis different from the positive charge carrier, the liposomes having anisoelectric point of between 4 and
 8. 2. The amphoteric liposomes ofclaims 1, wherein the liposomes have an isoelectric point of between 5and
 7. 3. Amphoteric liposomes, wherein the liposomes comprise at leastone amphoteric charge carrier, the amphoteric charge carrier having anisoelectric point of between 4 and
 8. 4. The amphoteric liposomes of thepreceding claim, wherein the amphoteric charge carrier has anisoelectric point of between 5 and
 7. 5. Amphoteric liposomes, whereinthe liposomes comprise at least one amphoteric charge carrier and atleast one anionic and/or cationic charge carrier.
 6. Amphotericliposomes of claim 5, wherein the liposomes have an isoelectric point ofbetween 5 and
 7. 7. The amphoteric liposomes of one of the claims 1 to6, wherein the liposomes comprise a neutral lipid, selected from thegroup consisting of phosphatidyl choline, phosphatidyl ethanolamine,cholesterol, tetraether lipid, ceramide, sphigolipid and/or diacrylglycerol.
 8. The amphoteric liposomes of one of the preceding claims,wherein the liposomes have an average size of between 50 and 1000 nm,preferably between 70 and 250 nm and particularly between 60 and 130 nm.9. The amphoteric liposomes of one of the preceding claims, wherein theliposomes comprise an active ingredient.
 10. The amphoteric liposomes ofthe preceding claim, wherein the active ingredient is a protein, apeptide, a DNA, an RNA, antisense nucleotide and/or a decoy nucleotide.11. The amphoteric liposomes of one of the preceding claims, wherein atleast 80 percent of the active ingredient is in the interior of theliposome.
 12. A method for charging liposomes with active ingredients ofclaims 1 to 11, wherein a defined pH is used for the encapsulation and asecond pH is used for separating the material, which has not been bound.13. The method for charging liposomes with active ingredient of claims 1to 11, wherein the liposomes are permneabilized and closed off at adefined pH.
 14. The use of liposomes of one of the claims 1 to 11 forproducing nanocapsules.
 15. The use of liposomes of one of the claims 1to 11 for producing release systems in diagnostics.
 16. The use ofliposomes of one of the claims 1 to 11 for transporting and/or releasingactive ingredients.
 17. The use of liposomes of one or claims 1 to 11 asa sustained-release formulation and/or as a circulating depot.
 18. Theuse of liposomes of one of the claims 1 to 11 for intravenous orperitoneal application.
 19. The use of liposomes of one of the claims 1to 11 as vector for the in vivo, in vitro and ex vivo transfection ofcells.